A successful virus infection usually involves entry into the cell; uncoating, expression and replication of the genome; assembly and release of infectious virus particles; and defense against specific and non- specific host immune mechanisms. For this reason, some viral genes are required even for replication in tissue culture cells whereas other are only advantageous during animal infections. In a 4,500 base pair segment of the vaccinia virus genome, we found three genes that are homologous to the eukaryotic genes profilin (an actin binding protein), 3-beta- hydroxysteroid dehydrogenase, and Cu-Zn superoxide dismutase. The role of the profilin homolog was examined by deleting the gene and characterizing the properties of the mutant. Surprisingly, the mutant was not defective in intracellular virus movement, formation of specialized microvilli or release of mature infectious virions. By contrast, deletion of another gene, encoding a protein of 37,000 Daltons that is a component of the outer envelope of vaccinia virus, resulted in a mutant that was defective in formation of extracellular virus and virus spread but produced normal amounts of infectious intracellular virus. Although the roles of non- essential genes can be studied by deletion mutagenesis, other methods are required fore essential genes. We have used the regulatory elements of the Escherichia coli lac operon to construct a conditional lethal inducer- dependent virus. In this manner, we demonstrated the essentially of an 11,000 Dalton core protein.